Field of the Invention
This invention is in the field of medical therapy. In particular, the invention relates to methods for treating, preventing, or reversing liver disease or damage produced by chronic alcohol intake by administering at least one peroxisome proliferator activated receptor (SPAR) agonist.
Related Art
There is increasing evidence that ethanol desensitizes hepatocytes to the trophic actions of growth factors and cytokine networks. Indeed, rat hepatocytes exposed to ethanol in culture have a markedly reduced response to insulin and epidermal growth factor (EGF) stimulated DNA synthesis. Thus, short- and long-term ethanol exposure impairs hepatocyte DNA synthesis in vitro (Carter et al., Biochem. Biophys. Res. Commun. 128:767 (1985); Carter et al., Alcohol Clin. Exp. Res. 12:555 (1988)) and the ability of the liver to regenerate after partial hepatectomy (Diehl et al., Gastroenterology 99:1105 (1990); Diehl et al., Hepatology 16:1212 (1992); Wands et al., Gastroenterology 77:528 (1979)). The precise molecular mechanism(s) by which ethanol inhibits hepatocyte proliferation are poorly understood. Liver regeneration is regulated by several growth factors and cytokines of which tumor necrosis factor (TNF)-α, EGF, transforming growth factor alpha (TGF-α), interleukin (IL)-6, hepatocyte growth factor (HGF), and insulin are believed to be most important (Michalopoulos et al., Science 276:60 (1997); Pistoi et al., FASEB J. 10:819 (1996); Fausto, J. Hepatol. 32:19 (2000). In this regard, previous studies suggested that ethanol interferes with signal transduction cascades activated by HGF (Saso et al., Alcohol Clin. Exp. Res. 20:330A (1996)), EGF (Zhang et al., J. Clin. Invest. 98:1237 (1996); Higashi et al., J. Biol. Chem. 266:2178 (1991); Saso et al., Gastroenterology 112:2073 (1997)), or TNF-α (Akerman et al., Hepatology 17:1066 (1993)) and with Ca2+-mediated signals in hepatocytes (Sun et al., Mol. Endocrinol. 11:251 (1997)). Furthermore, chronic ethanol consumption disturbs G-protein expression and inhibits cyclic AMP-dependent signaling in regenerating rat liver (Diehl et al., FASEB J. 10:215 (1996); Hoek et al., FASEB J. 6:2386 (1992)). Other signal transduction pathways are also adversely influenced by in vivo exposure to ethanol, as shown by several investigators. For example, G protein coupling to the EGF receptor is impaired (Zhang et al., Biochem. Pharmacol. 61:1021 (2001)); TNF-α-induced expression of NFκβ and JNK following partial hepatectomy is altered (Diehl, Clin. Biochem. 32:571 (1999)); and activation of p42/44, MAPK, p38 MAPK and JNK is reduced by acute or chronic ethanol exposure (Chen et al., Biochem. J. 334-669 (1998)).
Since hepatocyte growth factors such as EGF, TGF-α, HGF, and insulin activate receptor tyrosine kinases, the effects of ethanol on signal transduction as mediated through tyrosine phosphorylation of their intracellular substrates have been examined. Previous work established the biologic relevance of this pathway since ethanol is a potent inhibitor of DNA synthesis (Carter et al., Biochem. Biophys. Res. Commun. 128:767 (1985); Diehl et al., Gastroenterology 99:1105 (1990); Wands et al., Gastroenterology 77:528 (1979); Duguay et al., Gut 23:8 (1982)), and both insulin (Sasaki et al., Biochem. Biophys. Res. Commun. 199:403 (1994)) and cyclic AMP-mediated signal transduction (Diehl et al., Hepatology 16:1212 (1992)). These effects of ethanol may be mediated by uncoupling of insulin signal transduction pathways that are involved in mitogenesis (Sasaki et al., Biochem. Biophys. Res. Commun. 199:403 (1994)). Indeed, IRS-1-mediated signaling plays a critical role in regulating hepatocyte growth in the adult liver (Ito et al., Mol. Cell. Biol. 16:943 (1996); Nishiyama et al., Biochem. Biophys. Res. Commun. 183:280 (1992); Sasaki et al., J. Biol. Chem. 268:3805 (1993); Tanaka et al., J. Biol. Chem. 271:14610 (1996); Tanaka et al. Hepatology 26:598 (1997); Tanaka et al., Cancer Res. 56:3391 (1996); Tanaka et al., J. Clin. Invest. 98:2100 (1996)). It has been demonstrated in a transgenic (Tg) mouse model in which IRS-1 was overexpressed under the control of an albumin promoter, liver mass was 20-30% greater in the Tg mice relative to the non-Tg control mice, and that this difference was maintained throughout adult life (Tanaka et al. Hepatology 26:598 (1997)). This effect of IRS-1-overexpression was associated with increased hepatocyte DNA synthesis, and constitutive activation of the PI3K and Ras/Erk MAPK cascades. In other models, expression of antisense IRS-1 RNA or microinjection of IRS-1 antibodies inhibited insulin-stimulated DNA synthesis and growth (Rose et al., Proc. Natl. Acad. Sci. USA 91:797 (1994); Waters et al., J. Biol. Chem. 268:22231 (1993)). Similarly, a dominant/negative IRS-1 mutant was found to block insulin and IGF-I stimulated cell proliferation (Tanaka et al., J. Clin. Invest. 98:2100 (1996)). Furthermore, recent investigations demonstrated that activation of signaling through IRS-1 is essential for DNA synthesis and cell cycle progression, and that IRS-1 has a direct role in cellular transformation (Ito et al., Mol. Cell. Biol. 16:943 (1996); Tanaka et al., J. Biol. Chem. 271:14610 (1996); Tanaka et al., Cancer Res. 56:3391 (1996); Tanaka et al., J. Clin. Invest. 98:2100 (1996)). The effects of IRS-1 overexpression on cell growth seem to depend on constitutive activation of the mitogenic signal transduction cascades (Ito et al., Mol. Cell. Biol. 16:943 (1996); Nishiyama et al., Biochem. Biophys. Res. Commun. 183:280 (1992); Sasaki et al., J. Biol. Chem. 268:3805 (1993); Tanaka et al., J. Biol. Chem. 271:14610 (1996); Tanaka et al. Hepatology 26:598 (1997); Tanaka et al., Cancer Res. 56:3391 (1996); Tanaka et al., J. Clin. Invest. 98:2100 (1996)). Taken together these studies emphasize the importance of insulin/IGF-I signaling in liver growth and the potential adverse effect of insulin resistance on the hepatic repair process.
Insulin, a well-known hepatotrophic factor, acts through the insulin receptor (IR) to play an important role in liver growth and metabolism (Khamzina et al., Mol. Biol. Cell 9:1093 (1998); White, Recent Prog. Horm. Res. 53:119 (1998)). Upon binding to the IRα-subunit, the IRβ-subunit is autophosphorylated on tyrosyl residues, resulting in enhanced receptor tyrosyl kinase activity (White, Recent Prog. Horm. Res. 53:119 (1998)). IRS-1 is a major intracellular substrate for IR tyrosine kinase (Sun et al., Nature 352:73 (1991)). Tyrosyl-phosphorylated motifs located within the C-terminal region of IRS-1 protein (Myers et al., Trends Biochem. Sci. 19:289 (1994)) transmit signals downstream through interactions with SH2-containing molecules, including the p85 regulatory subunit of phosphatidylinositol 3′-kinase (PI3K) (Backer et al., EMBO J. 11:3469 (1992); Myers et al., Proc. Natl. Acad. Sci. USA 89:10350 (1992)), growth factor receptor-bound protein 2 (Grb2) (Skolnik et al., Science 260:1953 (1993)), phospholipase Cβ (PLCγ) (White, Recent Prog. Horm. Res. 53:119 (1998)), and tyrosyl phosphatase SHP2/Syp (Sun et al., Mol. Cell. Biol. 13:7418 (1993)). These binding events are critical to the activation of specific signaling pathways such as the Ras/Raf/MAPKK/MAPK cascade. Another major pathway of interest involves the binding of the p85 subunit of PI3K to the 613YMPM and 942YMKM motifs of IRS-1 (Backer et al., EMBO J. 11:3469 (1992); Myers et al., Proc. Natl. Acad. Sci. USA 89:10350 (1992)) which promotes cell survival by activating Akt/protein kinase B (PKB) (Dudek et al., Science 275:661 (1997); Eves et al., Mol. Cell. Biol. 18:2143 (1998)). PLCγ, acting on membrane phospholipids, produces second messengers, inositol phosphates and diacylglycerol, involved in control of intracellular Ca2+ levels, and protein kinase C activity (Carpenter et al., Exp. Cell Res. 253:15 (1999); Sekiya et al., Chem. Phys. Lipids 98:3 (1999)). The N-terminal sequences of IRS-1 contain three important functional domains that mediate signaling: one has been identified as a pleckstrin homology (PH) region (Musacchio et al., Trends Biochem. Sci. 18:343 (1993)), and two others as phosphotyrosine binding (PTB) domains (Sun et al., Nature 377:173 (1995); Gustafson et al., Mol. Cell. Biol. 15:2500 (1995)). The PH domain mediates IRS-1 interactions with the Tyk-2 Janus tyrosine kinase (Platanias et al., J. Biol. Chem. 271:278 (1996)) and may mediate cross-talk between IRS-1 and G protein (Touhara et al., J. Biol Chem. 269:10217 (1994)) or phospholipids (Harlan et al., Nature 371:168 (1994)) signaling.
One of the most important downstream signaling molecules engaged by IRS proteins is PI3K, which phosphorylates phosphoinositides at the D-3 position (Carpenter et al., Mol. Cell. Biol. 13:1657 (1993); Dhand et al., EMBO J. 13:511 (1994)). These phospholipids activate phosphoinositide-dependent kinase-1 (PDK-1), which phosphorylates Akt, a serine kinase (also referred to as PKB) (Kandel et al., Exp. Cell Res. 253:210 (1999); Franke et al., Cell 81:727 (1995); Burgering et al., Nature 376:599 (1995)) and activates Akt kinase. Many trophic factors, including insulin, utilize the PI3K→PDK1 pathway to increase Akt activity. Lipid products of PI3K can also activate phosphoinositide-dependent kinases, small G-proteins and protein kinase C (Avruch et al., Mol. Cell. Biochem. 182:31 (1998); Le Good et al., Science 281:2042 (1998); Zheng et al., J. Biol. Chem. 269:18727 (1994)) signal transduction molecules. In addition, recent evidence suggests that PI3K can directly control the activities of individual components of the Ras/Raf/MAPK pathway (Chaudhary et al., Curr. Biol. 10:551 (2000)).
Several downstream targets of PI3K, such as Akt, play a critical role in regulating transcription and cell fate. These effects require delicate and accurate balancing signals necessary for survival and programmed cell death (apoptosis) (Burgering et al., Nature 376:599 (1995); Franke et al., Cell 88:435 (1997)). Insulin induces Akt phosphorylation on two sites (Thr308 and Ser473), thereby activating Akt kinase in a PI3K-dependent manner (Carpenter et al., Mol. Cell. Biol. 13:1657 (1993)). GSK3β, a ubiquitously expressed serine/threonine protein kinase, is another key element of the PI3K/Akt pathway (Kandel et al., Exp. Cell Res. 253:210 (1999); Avruch et al., Mol. Cell. Biochem. 182:31 (1998)). High levels of GSK3β activity promote apoptosis. Akt kinase promotes survival and inhibits apoptosis in part by phosphorylating GSK3 at Ser 9/21 (Pap et al., J. Biol. Chem. 273:19929 (1998)), which deactivates the kinase and inhibits GSK3β activity (Srivastava et al., Mol. Cell. Biochem. 182:135 (1998); Cross et al., Nature 378:785 (1995)). Biologic activity of GSK3β is of particular interest because this kinase may regulate the level and function of aspartyl (asparaginyl) β-hydroxylase (AAH), which is an insulin-regulated, ethanol-sensitive gene that is a downstream target of IRS-1 signaling and an important mediator of cell migration.
Akt-dependent phosphorylation of the pro-apoptotic protein BAD, a member of the Bcl-2 family, is another key event in the cell survival process. BAD and phospho-BAD levels participate in regulating the balance between apoptosis and insulin-induced cell survival. The BAD Ser112 site-specific kinase is a mitochondrial membrane-localized cAMP-dependent protein kinase (PKA) (Harada et al., Mol. Cell 3:413 (1999)), that promotes a subcellular kinase-substrate interaction whereby an outer mitochondrial membrane protein, A-kinase anchoring protein, tethers the PKA holoenzyme to the organelle where BAD is active. Upon exposure to a survival factor such as insulin, the localized catalytic subunit of PKA phosphorylates mitochondrial-based BAD on Ser112. Phosphorylation of BAD at Ser112 and Ser136 inactivates and displaces BAD from binding to Bcl-2, causing BAD to translocate to the cytosol. This process inhibits apoptosis and promotes cell survival (Zha et al., Cell 87:619 (1996)).
There is now compelling evidence that under some circumstances, Akt activity can inhibit apoptosis by regulating transcription factors that control the expression of cell death genes. Recently, it was demonstrated that Akt regulates the FKHRL1 gene, a member of the Forkhead family of transcription factors. When Akt is activated by insulin/IGF-I stimulation, presumably via the IRS-1 signal transduction pathway, it phosphorylates FKHRL1 and promotes its association with 14-3-3 chaperone proteins, resulting in retention of FKHRL1 transcription factor in the cytoplasm. In contrast, reduced phosphorylation of FKHRL1 by Akt can lead to its nuclear translocation and subsequent activation of target genes. In this regard, one of the most important targets of FKHRL1 is the Fas ligand (L) gene, which contains 3 consensus sequences for FKHRL1 DNA binding. Binding of FKHRL1 to the Fas L promoter region increases Fas L gene expression and promotes cell death (Brunet et al., Curr. Opin. Neurobiol. 11:297 (2001)). Thus, inhibition of Akt signaling induces Fas L expression (Suhara et al., Mol. Cell. Biol. 22:680 (2002)). In this regard, the findings in previous experiments suggest that acute or chronic ethanol consumption may lead to up-regulated Fas L expression in rats and mice (Deaciuc et al., Alcohol Clin. Exp. Res. 23:349 (1999); Zhou et al., Am. J. Pathol. 159:329 (2001); Deaciuc et al., Hepatol. Res. 19:306 (2001); Castaneda et al., J. Cancer Res. Clin. Oncol. 127:418 (2001)), although the mechanism(s) has not yet been determined. It has been suggested that Fas L gene upregulation in the setting of chronic ethanol abuse may occur via reduced Akt dependent phosphorylation of Forkhead transcription factors, and this process activates programmed cell death mechanisms in hepatocytes. Therefore, it is important to recognize the biologic consequences of ethanol-induced alterations in both IRS-1-dependent and IRS-1-independent signaling mechanisms as they pertain to hepatocyte proliferation and survival.
There is direct experimental evidence that ethanol reduces PI3K activity in the liver by an IRS-1 dependent pathway. However, consideration of other potential mechanisms by which PI3K might be inhibited by ethanol led to examination of PTEN expression in the liver. The rationale for these experiments is that PTEN has emerged as a key negative regulator of PI3K activity (Yamada et al., J. Cell Sci. 114:2375 (2001); Seminario et al., Semin. Immunol. 14:27 (2002); Leslie et al., Cell Signal. 14:285 (2002); Maehama et al., Annu. Rev. Biochem. 70:247 (2001); Comer et al., Cell 109:541 (2002)). The PTEN molecule has multiple conserved domains such as a C2 phospholipid binding domain, two PEST regions, a PDZ binding domain, and a PIP2 binding motif. Initially described as a tumor suppressor gene, it has now been determined that the major substrates (in vivo) are PI (3, 4, 5) P3 and PI (4, 5) P2. The presence of a point mutation that abrogates only the lipid phosphatase domain of PTEN (G-129-E) is sufficient to produce a PTEN −/− phenotype including loss of tumor suppression. On the other hand, overexpression of PTEN blocks IRS-1 tyrosyl phosphorylation and IRS-1/Grb-2/SOS complex formation without interfering with tyrosyl phosphorylation of the insulin receptor. The net effect within the cell is to inhibit MAPK activation, cell cycle progression, and proliferation (Weng et al., Hum. Mol. Genet. 10:605 (2001)). These findings suggest that PTEN negatively regulates signaling pathways involved in hepatocyte proliferation.
Thus, it is of great interest with respect to ethanol effects on the liver that PTEN dephosphorylates and inhibits PI3K function (Dahia et al., Hum. Mol. Genet. 8:185 (1999); Maehama et al., Trends Cell. Biol. 9:125 (1999); Li et al., Cancer Res. 57:2124 (1997)). Conversely, inactivation of PTEN enhances PI3K function and promotes membrane recruitment of Akt leading to increased Akt phosphorylation and Akt kinase activity (Kandel et al., Exp. Cell Res. 253:210 (1999); Maehama et al., Trends Cell. Biol. 9:125 (1999)). Therefore, low levels of PTEN increase Akt kinase activity and promote growth and survival, whereas high levels of PTEN inhibit PI3K/Akt and enhance transmission of apoptotic signals, as well as inhibit cell proliferation. There is significantly increased PTEN expression and phosphatase activity in liver tissue derived from chronic ethanol-exposed rats relative to control rats. This phenomenon may be due to transcriptional, post-translational or both mechanisms. In this regard, PTEN expression and phosphatase activity are negatively regulated by phosphorylation (Torres et al., J. Biol. Chem. 276:993 (2001)), and suggest that PTEN levels may be controlled by insulin-induced phosphorylation in primary hepatocyte cultures, and that ethanol exposure enhances the biologic activity and levels of the protein by altering phosphorylation of the C-terminal region of the molecule. It is noteworthy that in ethanol-exposed liver tissue the magnitude of increased PTEN expression was paralleled by enhanced GSK-3β activity, consistent with the expected inhibitory effects of PTEN on downstream signaling through PI3K. This observation suggests that ethanol may have a major effect in the liver on both programmed cell death as well as proliferative pathways through modulation of PTEN expression.
The biologic significance as it applies to human disease is that ethanol may adversely affect specific signaling cascades related to both hepatocyte proliferation and survival as regulated by insulin and insulin like growth factors (IGF-I and IGF-II) and IRS-1-dependent and IRS-1-independent signal transduction cascades. There are ethanol effects on the Fas system, which appears to be linked to impaired PI3K/Akt signaling. Proliferative and survival pathways through IRS-1 and PI3K are strikingly altered by chronic ethanol exposure in vivo, and the Fas signaling pathway may contribute to hepatocyte apoptosis both in vitro and in vivo. Indeed, there is accumulating evidence that ethanol upregulates Fas L expression, and may play an important role in hepatocyte injury observed in alcoholics with liver disease, but the molecular mechanism(s) of Fas Receptor (R)/Fas L alterations by ethanol remain to be established (Benedetti et al., J. Hepatol. 6:137 (1988); Natori et al., J. Hepatol. 34:248 (2001); Goldin et al., J. Pathol. 171:73 (1993); Nanji, Semin. Liver Dis. 18:187 (1998); Higuchi et al., Hepatology 34:320 (2001)).
Another adverse effect of chronic ethanol consumption on the liver relates to oxidative stress as manifested by lipid peroxidation and DNA damage. In this regard, ethanol exposure to the liver increases cellular production of reactive oxygen species (ROS) that includes H2O2. When this occurs, the anti-oxidant defenses are inadequate, hepatocyte viability decreases and liver damage is produced (Sohn et al., J. Neurol. Sci. 162:133 (1999); Tanaka et al., J. Clin. Invest. 103:341 (1999); Diehl et al., Am. J. Physiol. Gastrointest. Liver Physiol. 288:1 (2005)). In this setting, there is increased liver inflammation that leads to chronic disease such as fatty liver, alcoholic hepatitis, cirrhosis and in some cases the development of hepatocellular carcinoma. Therefore chronic ethanol consumption has two major adverse effects on the liver, namely the induction and perpetuation of liver injury through oxidative stress, mitochondrial damage, upregulation of programmed cell death pathways, DNA damage and inhibition of the hepatic repair process (i.e. liver regeneration) principally through the insulin/IGF-I signal transduction cascades and it is one of the major causes of human liver disease throughout the world.
The magnitude of this problem is illustrated by the following: approximately 67% of adults consume alcohol and 14 million Americans meet the criteria for alcohol abuse and/or dependence. In this context, alcoholic liver disease (ALD) affects more than 2 million Americans and many more worldwide. The clinical consequences are that 40% of individuals with ALD die from cirrhosis of the liver. Thus, there is a real need to discover ways to prevent or treat these dreaded complications of ethanol abuse.